Chicken suffering from leucocytozoonosis show symptoms of a pale crown, excretion of green feces, loss of the glossy appearance of the feather, internal hemorrhage, malaise, dramatic reduction in egg production, and even death. The etiologic agent of leucocytozoonosis is Leucocytozoon calleryi, a protozoan parasite of the chicken and the mosquito, Cullicoid arakawae. When the mosquito carrying L. calleryi bites the chicken for the blood, the sporozoite of L. calleryi in the salivary gland of the mosquito is injected with the saliva into the chicken. The sporozoite migrates to the internal organs such as the liver and spleen, where it proliferates, emerges as schizonts and enters into the blood. The schizont infects the red blood cells, proliferates, and transforms into merozoites, which in turn infects other red blood cells, and proliferates, producing more merozoites. This cycle is repeated several times. Thus, the red blood cells are repeatedly infected and destroyed, and as a result, the chicken becomes anemic and loses the redness of the crown. The merozoites then become gametocytes, which enter the mosquito when the mosquito bites the diseased chicken. The gametocytes in the mosquito mate to form zygotes, which, as they mature, further migrate to the salivary gland, where they transforms into sporozoites, to complete the life cycle.
Many drugs have been used for the treatment of leucocytozoonosis, but most are ineffective except pyrimethamine. Unfortunately, pyrimethamine is not only inducing resistant strains but also carcinogenic and can remain in the egg, so that it would be a health hazard for humans. Therefore, pyrimethamine is prohibited from using in the chicken by the authority. There are no effective drugs available for the treatment of leucocytozoonosis in chicken. Leucocytozoonosis causes damage to the chicken industry and consequently, the damage induces great economic loss. Chicken leucocytozoonosis must be controlled.
An alternative and better approach to deal with a disease is prevention, and the most effective and economical approach is to use a vaccine. There are various types of vaccine, depending on the purpose. Some vaccines induce humoral immunity, i.e., specific antibody, and others induce cellular immunity, i.e., leucocytes. Since the etiologic agent of chicken leucocytozoonosis is a protozoon, Leucocytozoon, the immunization effect of antibody is limited. Furthermore, in order to prepare a vaccine that induces humoral immunity, a large amount of Leucocytozoon must be collected first, and mass production of Leucocytozoon is problematical because it is tedious and difficult. To collect schizonts, merozoites or gametocytes, of Leucocytozoon, for example, a huge volume of infected chicken blood during a certain diseased period must be collected first, and these different stages of Leucocytozoon are then isolated and purified from the blood. To gather sporozoites, one must grow L. calleryi, the chicken mosquito, in a large scale, allow them to bite diseased chicken in an appropriate period, and then isolate sprozoites from the salivary glands of these mosquitoes. In either case, the diseased chicken in a certain disease period must be used, because the protozoa in various stages and sporozoite only appear, respectively, in a certain phase of the diseased chicken. Thus, these materials are very difficult to prepare. Furthermore, a vaccine that is against the protozoa in a stage, such as merozoite, will not be effective against the protozoa in other stages, such as sporozoite, because of different antigenecity. Such vaccine, consequently, will not be effective against leucocytozoonosis.
A better approach to protect chicken against chicken leucocytozoonosis is the use of vaccine that can induce cellular immunity. Several considerations must be taken into account in the construction of such vaccine. The first is to determine the target of the vaccine. As described above, there are at least three forms of leucocytozoon in the blood, which means several different antigens. Furthermore, each form has unique and varied antigens. Immunity against a form will not be effective against another form. Thus, a generalized vaccine effective against all leucocytozoon stages in the blood is difficult to make.
When the mosquito injects the leucocytozoon into the chicken it is in the form of sporozoite, which remains so until it reaches the internal organs such as the liver. In contrast to varied antigenecities in the blood, the surface of a sporozoite is largely made up of one antigen, called circumsporozoite (CS) protein, which is very immunogenic. Consequently, CS protein would be a very good vaccine candidate. The next thing one must consider is where to induce the immunity. The internal organs such as the liver are certainly the main candidate location for this, since the destination of a sporozoite is the internal organs. How can the immunity be induced in the liver, or other internal organs? If vaccine could reach the target organs, the immunity could be induced at that organ.
For this purpose, we chose Salmonella enterica serovar Typhimurium as the vaccine carrier. The portal of entry of Salmonella such as Typhimurium is gastrointestinal. Typhimurium, furthermore, can cause systemic infection, i.e., it can migrate to the internal organs such as the liver and spleen. Typhimurium is a pathogen, and therefore, one must detoxify Typhimurium (make, it avirulent) before it can be used as a vaccine carrier. However, an avirulent Typhimurium must still retain the ability to migrate to the internal organs, where the immunity must be induced, and must be able to survive, in vivo, for a short period of time. We have obtained such avirulent Typhimurium.
The target of the vaccine is leucocytozoon and therefore, the vaccine must be able to induce the immunity against it. Thus, the vaccine must carry the antigen of sporozoite. To allow a vaccine carrier to carry the antigen, the carrier must contain the gene of the antigen and express it. We are able to clone the CS gene and successfully transfer the CS gene into the vaccine carrier, which expresses the antigen. This is the vaccine.